Peptide mimotope to mycotoxin deoxynivalenol and uses thereof

ABSTRACT

The present invention provides a peptide mimotope of the non-peptide mycotoxin deoxynivalenol. In particular, the peptide mimotope competes with deoxynivalenol for binding to a monoclonal antibody and is antagonistic to the inhibitory effects of deoxynivalenol on in vitro protein synthesis. The present invention also provides a method that uses the peptide mimotope to determine whether corn, grains or mixed feed is contaminated with fungi that produces deoxynivalenol. The present invention further provides transgenic plants resistant to deoxynivalenol.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional SerialNo. 60/146,643 filed Jul. 30, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was supported by U.S. Department of AgricultureResearch National Research Initiative grant 9702545 and Public HealthService grant E5-03358. The United States has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] (1) Field of the Invention

[0004] The present invention relates to a peptide mimotope of thenon-peptide mycotoxin deoxynivalenol. In particular, the peptidemimotope competes with deoxynivalenol for binding to a monoclonalantibody, is antagonistic to the inhibitory effects of deoxynivalenol onin vitro protein synthesis, and does not elicit antibodies in mice thatrecognize the deoxynivalenol. The present invention also relates to amethod that uses the peptide mimotope to determine whether corn, grainsor mixed feed is contaminated with fungi that produces deoxynivalenol.The present invention further relates to transgenic plants resistant todeoxynivalenol.

[0005] (2) Description of Related Art

[0006] Deoxynivalenol (DON) or vomitoxin or dehydronivalenol is12,13-epoxy-3,7,15-trichothec-9-en-8-one, which is a mycotoxin of the12,13-epoxy-trichothecenes class of sesquiterpene mycotoxins. It isproduced primarily by the fungus Gibberella zeae (Schwein.) Petch(anamorph=Fusarium graminearum Schwabe), which infects corn, smallgrains and mixed feeds (Hart et al., J. Agric. Food Chem. 31: 657-659(183); Hart et al., Plant Dis. 66: 1133-1135 (1982); Neish et al., Can.J. Plant Sci. 61: 811-815 (1981)). At the cellular level, the primarytoxic effect of DON is inhibition of protein synthesis by binding to the60S ribosomal subunit, which interferes with peptidyltransferase(Betina, Chem. Biol. Interact. 71: 105-146 (1989); Weber et al.,Biochem. 31: 9350-9354 (1992)). DON can cause anorexia and emesis inanimals (Scott et al. Proc. natl. Acad. Sci. USA 89: 5398-5402 (1992)).Other toxic effects of DON include skin irritation, hemorrhaging,hematological changes, human lymphocyte blastogenesis impairment,radiomimetic effects, apoptosis and immunotoxicity (Scott et al. ibid.).

[0007] DON is primarily found as a contaminant in grains that areinfected with the above fungi. It has also been implicated as a chemicalwarfare agent. Currently, the only means for eliminating DON from humanand animal foodstuffs is to detect DON in food and to remove anycontaminated foodstuffs from the food supply. Immunoassays offer severaladvantages compared to other analytical methods for detecting DON infoodstuffs. Following the development of the first monoclonal antibodyto DON (Casale et al., J. Agric. Food Chem. 36: 663-668 (1988)),immunological methods, primarily enzyme-linked immunosorbant assay(ELISA), have been widely used for detection of DON (Pestka et al., FoodTechnol. 49: 120-128 (1995)). An immunoassay for trichothecenes such asDON is disclosed in U.S. Pat. No. 4,879,248 to Chu et al. and kitcomprising the immunoassay is disclosed in U.S. Pat. No. 5,118,612 toChu et al. The immunoassay and kit are either radio immunoassays (RIA)or enzyme-linked immunosorbant assay ELISA based on a competitivecontrol that is DON. These immunological assays have advantages whichinclude high specificity, ease of use, facile sample preparation, andgood sensitivity.

[0008] The disadvantages of these immunoassays is that they require theuser to handle purified DON which poses a toxicity risk to the user. Inaddition, chemical conjugation of DON to a carrier protein or an enzymehas low efficiency because it involves extensive modification andblocking stages and causes substantial bridge-group interferences andun-wanted cross-reactions (Casale et al., ibid.: Pestka et al., ibid.;Yuan et al., Appl. Environ. Microbiol. 63: 263-269 (1997)). Furthermore,DON is poorly immunogenic and when DON is conjugated to a carrierprotein, it's immunogenicity is only marginally enhanced.

[0009] Therefore, it is desirable that an alternative to DON bedeveloped. Preferably, the DON alternative would be non-toxic to theuser, not require conjugation to a protein, and be highly immunogenic.

SUMMARY OF THE INVENTION

[0010] The present invention provides a peptide mimotope of thenon-proteinaceous mycotoxin deoxynivalenol (DON). In particular, thepeptide mimotope competes with DON for binding to a monoclonal antibodyagainst the DON, is antagonistic to the inhibitory effects of DON on invitro protein synthesis, and does not elicit antibodies in mice thatrecognize DON.

[0011] The peptide mimotope comprises amino acid sequence SWGPX₁PX₂ (SEQID NO: 6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acidor analog thereof. In particular species of the present invention, apeptide mimotope of DON is provided comprising the amino acid sequenceSWGPFPF (SEQ ID NO: 2), a peptide mimotope of DON comprising the aminoacid sequence SWGPLPF (SEQ ID NO: 4), or a peptide mimotope of DONcomprising the amino acid sequence SWGPFPFGGGSC (SEQ ID NO: 5). Thepeptide mimotope species are antagonistic to the inhibitory effects ofDON on in vitro protein synthesis. In a preferred embodiment, thepeptide mimotope is conjugated to a reporter for an immunological assaywherein the reporter is selected from the group consisting of alkalinephosphatase, horseradish peroxidase, or fluorescence molecule. Inanother preferred embodiment, the peptide mimotope is a part of apeptide or polypeptide. In particular, as a fusion polypeptide whereinthe polypeptide is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.

[0012] The present invention further provides a nucleic acid thatencodes the peptide mimotope of DON comprising an amino acid sequenceselected from the group consisting of SWGPLPF (SEQ ID NO: 2), SWGPFPF(SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5). In particularembodiments, the nucleic acid sequence is selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 3.

[0013] The present invention also provides a clone in a microorganismexpressing a peptide mimotope of DON comprising amino acid sequenceSWGPX₁PX₂ (SEQ ID NO: 6) wherein X₁ is L, F, or analog thereof and X₂ isany amino acid or analog thereof. In particular species, the peptidemimotope comprises an amino acid sequence selected from the groupconsisting of SWGPFPF (SEQ ID NO: 2), SWGPLPF (SEQ ID NO: 4), andSWGPFPFGGGSC (SEQ ID NO: 5). In particular, the peptide mimotopeexpressed by the clone is antagonistic to the inhibitory effects of DONon in vitro protein synthesis. For the clone expressing the peptidemimotope, the peptide mimotope is encoded by a nucleic acid in a plasmidor by a nucleic acid in a recombinant virus vector such as abacteriophage and the peptide mimotope can be expressed as an isolatedpeptide or as a part of a fusion polypeptide. Furthermore, themicroorganism containing the clone expressing the peptide mimotope canbe selected from the group consisting of bacteria and yeasts.

[0014] The present invention further provides a transgenic plantcontaining a nucleic acid that expresses a peptide mimotope of DON thatbinds to a monoclonal antibody against DON and is antagonistic to theinhibitory effects of DON on in vitro protein synthesis. In particular,the present invention provides a transgenic plant that expresses apeptide mimotope of DON comprising amino acid sequence SWGPX₁PX₂ whereinX₁ is L, F, or analog thereof and X₂ is any amino acid or analogthereof. In particular species, the amino acid sequence is selected fromthe group comprising SWGPFPF (SEQ ID NO: 2), SWGPLPF (SEQ ID NO: 4), andSWGPFPFGGGSC (SEQ ID NO: 5). Further, the peptide mimotope that isexpressed can be as an isolated peptide or as a part of a fusionpolypeptide.

[0015] The present invention also provides an improvement in a methodfor determining whether a sample contains DON which comprises providinga monoclonal antibody against the DON, reacting the monoclonal antibodywith the sample in a reaction mixture containing a labeled DON as acompetitor, and determining whether the sample contains DON, wherein theimprovement is providing as the competitor a peptide mimotope of DON. Inparticular, the peptide mimotope has amino acid sequence SWGPX₁PX₂ (SEQID NO: 6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acidor analog thereof. In particular species, the amino acid sequence isselected from the group consisting of SWGPFPF (SEQ ID NO: 2), SWGPLPF(SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5). Further, the peptidemimotope can be as an isolated peptide or as a part of a fusionpolypeptide.

[0016] The present invention also provides a method for determiningwhether a sample contains deoxynivalenol (DON) which comprises: (a)incubating in a reaction the sample, a monoclonal antibody against theDON, and a peptide mimotope which is a competitor of the DON for themonoclonal antibody; (b) detecting in the reaction a complex consistingof the DON bound by the monoclonal antibody and a complex formed by themimotope and monoclonal antibody; and (c) comparing an amount of each ofthe complexes wherein a decrease in the amount of the complex comprisingthe peptide mimotope indicates that the sample contains DON. Inparticular, the peptide mimotope comprises amino acid sequence SWGPX₁PX₂(SEQ ID NO: 6) wherein X₁ is L, F, or analog thereof and X₂ is any aminoacid or analog thereof. In particular species, the amino acid sequenceis selected from the group consisting of SWGPFPF (SEQ ID NO: 2), SWGPLPF(SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5). In a preferredembodiment of the method, the monoclonal antibody is produced byhybridoma cell line 6F5. The method further comprises the peptidemimotope which is conjugated to an enzyme selected from the groupconsisting of horseradish peroxidase and alkaline phosphatase; thepeptide mimotope conjugated to a fluorescent reporter; and the peptidemimotope wherein the amino acid of the peptide mimotope is conjugated toan enzyme selected from the group consisting of horseradish peroxidaseand alkaline phosphatase to make a fusion protein. Alternatively, thepeptide mimotope can be part of a fusion polypeptide wherein thepolypeptide is an enzyme that is used as a reporter enzyme inimmunoassays, in particular alkaline phosphatase or horseradishperoxidase.

[0017] The present invention also provides a kit for determining whethera sample contains deoxynivalenol (DON) comprising: (a) a monoclonalantibody against the DON; (b) a peptide mimotope of the DON thatcompetes with DON for binding to the monoclonal antibody; and (c)instructions for using the kit. In particular, the mimotope comprisesamino acid sequence SWGPX₁PX₂ (SEQ ID NO: 6) wherein X₁ is L, F, oranalog thereof and X₂ is any amino acid or analog thereof. In particularspecies, the peptide mimotope of the kit comprises an amino acidsequence selected from the group consisting of SWGPFPF (SEQ ID NO: 2),SWGPLPF (SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5). In thepreferred embodiment, the monoclonal antibody is produced by hybridomacell line 6F5. The kit further comprises the peptide mimotope which isconjugated to an enzyme selected from the group consisting ofhorseradish peroxidase and alkaline phosphatase; the peptide mimotopeconjugated to a fluorescent reporter; and a fusion protein wherein theamino acid sequence comprising the peptide mimotope is conjugated to anenzyme selected from the group consisting of horseradish peroxidase andalkaline phosphatase. Alternatively, the peptide mimotope can be part ofa fusion polypeptide wherein the polypeptide is an enzyme that is usedas a reporter enzyme in immunoassays, in particular alkaline phosphataseor horseradish peroxidase.

[0018] The present invention further provides a method for making aplant resistant to deoxynivalenol (DON) comprising introducing into theplant plant's genome a nucleic acid that encodes a peptide mimotope,which binds to a monoclonal antibody against DON and is antagonistic tothe inhibitory effects of DON on in vitro protein synthesis, which isoperably linked to a transcription promoter. In particular, the peptidemimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ ID NO: 6) whereinX₁ is L, F, or analog thereof and X₂ is any amino acid or analogthereof. In a particular species, the peptide mimotope comprises anamino acid sequence selected from the group consisting of SWGPFPF (SEQID NO: 2), SWGPLPF (SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5). Thepeptide mimotope can be expressed as an isolated peptide or as a part ofa fusion polypeptide.

[0019] The present invention also provides a method for treating anorganism exposed to deoxynivalenol (DON) comprising treating theorganism with a peptide mimotope of DON which is antagonistic to theinhibitory effects of DON on in vitro protein synthesis. In particular,the peptide mimotope comprises amino acid sequence SWGPX₁PX₂ (SEQ ID NO:6) wherein X₁ is L, F, or analog thereof and X₂ is any amino acid oranalog thereof. In a particular species, the peptide mimotope comprisesan amino acid sequence selected from the group consisting of SWGPFPF(SEQ ID NO: 2), SWGPLPF (SEQ ID NO: 4), and SWGPFPFGGGSC (SEQ ID NO: 5).The treatment may be given orally, topically, or intravenously.Furthermore, the treatment can comprise a peptide mimotope of DON, whichis a vaccine that elicits antibodies against DON. The vaccine can beadministered either as a therapeutic treatment to an animal or persondisplaying symptoms of exposure to DON or as a prophylactic treatment toprevent symptoms caused by a subsequent exposure to DON. The peptidemimotope can be the isolated peptide or as a part of a fusionpolypeptide.

OBJECTS

[0020] It is therefore an object of the present invention to provide apeptide mimotope of deoxynivalenol (DON) for use in immunological assaysfor detecting DON in a sample.

[0021] It is also an object of the present to provide a transgenic plantwhich is resistant to the affects of DON.

[0022] It is a further object of the present invention to provide amethod for treating an animal or person exposed to DON.

DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1A shows a two-dimensional representation of DON. Theasterisk indicates the site of conjugation of the carrier protein, e.g.,BSA, to DON.

[0024]FIG. 1B shows a two-dimensional representation of nivalenol, ananalog of DON whose three-dimensional structure is known.

[0025]FIG. 1C shows a three-dimensional structural model of the DONpeptide mimotope. The white spheres represent oxygen atoms, the whitecylinders represent nitrogen atoms, and the grey cylinders representcarbon atoms.

[0026]FIG. 1D shows a three-dimensional view of the crystallographicstructure of nivalenol (CSD entry: DUTJOR10. The white spheres representoxygen atoms, the white cylinders represent nitrogen atoms, and the greycylinders represent carbon atoms.

[0027]FIG. 1E shows a stereo view of the optimal PowerFit superpositionof the known nivalenol structure and the DON peptide mimotope structure.Nivalenol aligns with the peptide model main-chain atoms from residues 2to 5 (TrpGlyProPhe or WGPF) and partially overlaps the side chains ofTrp-2 and Pro-4. The white spheres represent oxygen atoms, the whitecylinders represent nitrogen atoms, and the grey cylinders representcarbon atoms.

[0028]FIG. 2 shows the competition between phage-displayed peptidemimotopes and DON for binding to immobilized mAB 6F5 in a CD-ELISA.Various concentrations of free DON competed with equal volumes ofphage-displayed peptides (at a constant concentration) for binding toimmobilized mAB 6F5. Bound phage peptide was detected withHRP-conjugated sheep anti-M13 IgG and then measured by absorbance.DON-HRP was included as a positive control.

[0029]FIG. 3A shows synthetic peptide C430 and DON competing withDON-HRP for binding to immobilized mAB 6F5.

[0030]FIG. 3B shows synthetic peptide C430 and DON competing withC430-HRP for binding to immobilized mAB 6F5.

[0031]FIG. 4 shows a CD-ELISA performed with DONPEP-AP fusion protein.Binding of the DONPEP-AP fusion protein to immobilized mAB 6F5 wasinhibited by free DON. Competition of free DON with DON-HRP was used asa control.

[0032]FIG. 5 shows the use of C430 HRP and DONPEP-AP in a DONimmunoassay (CD-ELISA) performed with wheat extract spiked with DON.Immulon-4 microtiter wells were coated with mAB 6F5 and DON-HRP was usedas a positive control.

[0033]FIG. 6 shows the specificity of antibody obtained fromC430-BSA-immunized mice. DON and C430 at various concentrations wereused to inhibit the binding of antisera to immobilized DONPEP.2 phage.Bound mouse antibodies were detected with HRP-conjugated goat anti-mouseIgG, and the amounts of these antibodies were measured by absorbance.

[0034]FIG. 7 shows the effects of DON (3.4 μM) and synthetic peptideC430 (3.4 μM) on protein synthesis in vitro with rabbit reticulocytelysate. The translation template was γ-globulin mRNA. The plus and minussigns indicate which reagents were added.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0035] All patents, patent applications, and literature references citedin this specification are hereby incorporated herein by reference intheir entirety. In case of conflict, the present description, includingdefinitions, will control.

[0036] To promote a better understanding of the present invention, thefollowing terms are defined.

[0037] The term “mimotope” means a molecule which has a conformationthat has a topology equivalent to the epitope of which it is a mimic.The mimotope binds to the same antigen-binding region of an antibodywhich binds immunospecifically to a desired antigen. Generally, amimotope will elicit an immunological response in a host that isreactive to the antigen to which it is a mimic.

[0038] The term “mimetic” is a related mimotope which means a moleculewhich competes with the antigen for binding to the antibody but whichdoes not elicit an antibody in a host that is reactive against theantigen. The present invention is a mimetic.

[0039] The term “monoclonal antibody” as used herein refers toantibodies produced by a single line of hybridoma cells all directedtowards one epitope on a particular antigen. A hybridoma is a clonalcell line that consists of hybrid cells formed by the fusion of amyeloma cell and a specific antibody-forming cell. In general,monoclonal antibodies are of mouse origin; however, monoclonal antibodyalso refers to a clonal population of an antibody made against aparticular antigen or epitope of an antigen produced by phage displaytechnology or method that is equivalent to phage display or hybrid cellsof non-mouse origin.

[0040] The term “antigen” as used herein refers to a substance whichstimulates production of antibody or sensitized cells during an immuneresponse. An antigen consists of one or more epitopes, each epitope ofwhich is capable of causing the production of an antibody against theparticular epitope.

[0041] The term “epitope” as used herein refers to an immunogenic regionof an antigen which is recognized by a particular antibody molecule. Ingeneral, an antigen will possess one or more epitopes, each capable ofbinding an antibody that recognizes the particular epitope.

[0042] Amino acids are represented herein by the single letter ortriplet code wherein alanine is A or Ala, arginine is R or Arg,asparagine is N or Asn, aspartic acid is D or Asp, cysteine is C or Cys,glutamine is Q or Gln, glutamic acid is E or Glu, glycine is G or Gly,histidine is H or His, isoleucine is I or Ile, leucine is L or Leu,lysine is K or Lys, methionine is M or Met, phenylalanine is F or Phe,proline is P or Pro, serine is S or Ser, threonine is T or Thr,tryptophan is W or Trp, tyrosine is Y or Tyr, and valine is V or Val.

[0043] The nucleotides are represented herein by A for adenosine, G forguanosine, C for cytosine, and T for thymidine.

[0044] The present invention provides peptide mimotopes of thenon-peptide toxin, deoxynivalenol (DON). DON has the two-dimensionalstructure shown in FIG. 1A. The peptide mimotopes have a topologicalstructure that mimics the three-dimensional structure of DON. As shownin FIG. 1E, the three-dimensional structure of nivalenol, an analog ofDON which has the two-dimensional structure shown in FIG. 1B, which canbe aligned along the peptide mimotope main chain atoms from amino acidresidue 2 to 5, i.e., TrpGlyPro (WGP). The close structural alignment issufficient to enable the peptide mimotopes to be recognized by ananti-DON monoclonal antibody, mAB 6F5, and effectively compete with DONfor binding to the anti-DON monoclonal antibody (FIGS. 2-5).

[0045] Monoclonal antibody mAB 6F5 from hybridoma cell line mAB 6F5 wasprepared as described in Casale et al., J. Agric. Food Chem. 36: 663-668(1988). Monoclonal antibody mAB 6F5 is available from Michigan StateUniversity and is commercially available from Neogen Corporation, 620Lesher Place, Lansing, Mich. 48912. Alternatively, hybridomas thatproduce mAB against DON can be prepared as taught in Casale et al., J.Agric. Food Chem. 36: 663-668 (1988). Hybridoma clones that producemonoclonal antibodies against DON are then screened with a labeledpeptide mimitope which enables those hybridoma clones that react againstthe peptide mimotope to be identified.

[0046] The toxic effect of DON is caused by its binding to a particularsite on the 60S ribosome, which inhibits 60S ribosome function, therebypreventing protein synthesis. Competition experiments between DON andthe peptide mimotopes indicate that the peptide mimotopes bind to thesame site on the 60S ribosome as DON (FIG. 7); however, in contrast towhen DON is bound, the peptide mimotopes do not inhibit the function ofthe 60S ribosome. Thus, the present invention is both a mimic of the DONepitope that is recognized by the anti-DON monoclonal antibody and acompetitor that binds to the same site on the 60S ribosome as DON, butwithout DON's inhibitory effect. Since there are few examples ofmimotopes of non-proteinaceous chemicals other than biotin orcarbohydrates, it was uncertain whether a peptide sequence could befound that would mimic DON. Therefore, it was unexpected that thepeptide mimotopes which bind to the monoclonal antibody against DONwould be antagonistic to the toxic effects of DON on protein synthesisindicating it was a mimetic.

[0047] In general, the present invention provides peptide mimotopes thathave an amino acid sequence of SWGPX₁PX₂ (SEQ ID NO: 6) wherein theamino acid sequence mimics the DON epitope that is bound by anti-DONmonoclonal antibody mAB 6F5 and wherein X₁ and X₂ is each any amino acidor analog thereof, preferably wherein X₁ is an amino acid or analogthereof which has a side chain that is a hydrogen or alkyl, mostpreferably, wherein X₁ is L, F, or analog thereof. The SWGP is theportion of the peptide that can be aligned with the structure ofnivalenol and mimics the nivalenol structure. In particular, the presentinvention provides a peptide mimotope that has the amino acid sequenceselected from the group consisting of SWGPFPF (SEQ ID NO: 2), which ispeptide DONPEP.2; SWGPLPF (SEQ ID NO: 4), which is peptide DONPEP.12;and, SWGPFPFGGGSC (SEQ ID NO: 5), which is peptide C430 comprising theDONPEP.2 amino acid sequence coupled to amino acid sequence GGGSC. Eachof the aforementioned peptide mimotopes are artificial peptide sequencesthat are not naturally produced in nature. The peptide mimotopes can bemade in vitro using any one of the peptide synthesis methods which arewell known in the art, e.g., Fmoc peptide synthesis chemistry. Forparticular applications it is desirable to produce the peptide mimotopein vivo; therefore, the peptide DONPEP.2 is encoded by the nucleic acidsequence 5-AGTTGGGGTCCTTTTCCGTTT-3 (SEQ ID NO: 1) and peptide DONPEP.12is encoded by nucleic acid sequence 5′-TCTTGGGGTCCGCTTCCTTTT-3′ (SEQ IDNO: 3). Producing the peptide mimotope in vivo is particularly desirablewhen the peptide mimotope is to be expressed as a part of a fusionpeptide or polypeptide. For example, the phage clones disclosed hereinexpress a chimeric polypeptide that contains the peptide mimotope aminoacid sequence within and covalently linked to the amino acid sequencefor the minor coat protein of phage M13, and DONPEP-AP disclosed hereinis a chimeric polypeptide wherein the peptide mimotope amino acidsequence is between and covalently linked to the amino acid sequence forthe minor coat protein signal sequence and the amino acid sequence foralkaline phosphatase. In general, chimeric fusion polypeptides,particularly large fusion polypeptides, are more economically producedin vivo than in vitro.

[0048] The peptide mimotope can be synthetically produced by chemicalsynthesis methods which are well known in the art, either as an isolatedpeptide or as a part of another peptide or polypeptide. Alternatively,the peptide mimotope can be produced in a microorganism which producesthe peptide mimotope which is then isolated and if desired, furtherpurified. The isolated or purified peptide mimotope can be used as acontrol or competitor in immunoassays for detecting DON in food samples,or because it competes with DON for binding to the 60S ribosome, theisolated or purified peptide mimotope can be used in therapies fortreating animals or humans exposed to DON. Thus, the peptide mimotopecan be produced in microorganisms such as bacteria, yeast, or fungi; ina eukaryote cells such as a mammalian or an insect cells; or, in arecombinant virus vector such as adenovirus, poxvirus, herpesvirus,Simliki forest virus, baculovirus, bacteriophage, sindbis virus, orsendai virus. Suitable bacteria for producing the peptide mimotopeinclude Escherichia coli, Bacillus subtilis, or any other bacterium thatis capable of expressing peptides such as the peptide mimotope. Suitableyeast types for expressing the peptide mimotope include, but is notlimited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida,or any other yeast capable of expressing peptides. Methods for using theaforementioned bacteria, recombinant virus vectors, eukaryote cells toproduce peptides are well known in the art.

[0049] To produce the peptide mimotope, the nucleic acid encoding thepeptide mimotope is in a plasmid and the nucleic acid is operably linkedto a promoter which effects the expression of the peptide mimotope in amicroorganism. Suitable promoters include, but are not limited to, T7phage promoter, T3 phage promoter, β-galactosidase promoter, and the Sp6phage promoter. Expression of the peptide mimotope in a microorganismenables the peptide mimotope to be produced using fermentationtechnologies which are used commercially for producing large quantitiesof peptides. Methods for isolating and purifying peptides are well knownin the art and include methods such as gel filtration, affinitychromatography, ion exchange chromatography, or centrifugation.

[0050] To facilitate isolation of the peptide mimotope, a fusionpolypeptide is made wherein the peptide mimotope is translationallyfused (covalently linked) to a heterologous polypeptide which enablesisolation by affinity chromatography. Preferably, a fusion polypeptideis made using one of the expression systems infra. For example, thenucleic acid sequence encoding the peptide mimotope is linked at eitherthe 5′ end or 3′ end to a nucleic acid encoding a heterologouspolypeptide. The nucleic acids are covalently linked in the proper codonreading frame to enable production of a fusion polypeptide wherein theamino and/or carboxyl terminus of the peptide mimotope istranslationally fused to the heterologous polypeptide which allows forthe simplified recovery of the fusion polypeptide. The fusionpolypeptide can also prevent the mimotope polypeptide from beingdegraded during purification. While the fusion polypeptide isefficacious, as shown by the results herein for the phage clones orDONPEP-AP, in some instances it can be desirable to remove theheterologous polypeptide after purification. Therefore, it is alsocontemplated that the fusion polypeptide comprise a cleavage site at thejunction between the peptide mimotope and the heterologous polypeptide.The cleavage site consists of an amino acid sequence that is cleavedwith an enzyme specific for the amino acid sequence at the site.Cleavage sites that are contemplated include, but are not limited to,the enterokinase cleavage site which is cleaved by enterokinase, thefactor Xa cleavage site which is cleaved by factor Xa, and the GENENASEcleavage site which is cleaved by GENENASE (GENENASE is a trademark ofNew England Biolabs, Beverly, Mass.). The following are methods forproducing the peptide mimotope as a fusion polypeptide or as an isolatedpeptide mimotope free of the heterologous polypeptide.

[0051] An example of a procaryote expression system for producing thepeptide mimotope is the Glutathione S-transferase (GST) Gene FusionSystem available from Amersham Pharmacia Biotech, Piscataway, N.J.,which uses the pGEX-4T-1 expression vector plasmid. The nucleic acidencoding the peptide mimotope is fused in the proper codon reading framewith the nucleic acid encoding the GST polypeptide. The GST polypeptideallows the rapid purification of the fusion polypeptide usingglutathione Sepharose 4B affinity chromatography. After purification,the GST can be removed by cleavage with a site-specific protease such asthrombin or factor Xa to produce the mimotope free of the GSTpolypeptide. The peptide mimotope free of the GST polypeptide isproduced by a second round of glutathione Sepharose 4B affinitychromatography.

[0052] Another method for producing the peptide mimotope is a methodwhich links in-frame the nucleic acid encoding the peptide mimotope to anucleic acid encoding polyhistidine, preferably encoding six histidineresidues, to produce a peptide mimotope-polyhistidine fusionpolypeptide. The polyhistidine allows purification of the fusionpolypeptide by metal affinity chromatography, preferably nickel affinitychromatography. To produce the peptide mimotope free of thepolyhistidine, a cleavage site such as an enterokinase cleavage site isfused in the proper reading frame between the codons encoding thepolyhistidine and the codons encoding the peptide mimotope. Thus, thepeptide mimotope free of the polyhistidine is made by removing thepolyhistidine by cleavage with enterokinase. A second round of metalaffinity chromatography which binds the free polyhistidine results inthe peptide mimotope free of the polyhistidine. The Xpress System,available from Invitrogen, Carlsbad, Calif., is an example of acommercial kit which is available for making and then isolatingpolyhistidine fusion polypeptides.

[0053] In a method further still, the pMAL Fusion and PurificationSystem available from New England Biolabs can be used to make a fusionpolypeptide wherein a maltose binding protein is fused to the peptidemimotope. The maltose binding protein facilitates isolation of thefusion polypeptide by amylose affinity chromatography. The maltosebinding protein can be linked to the peptide mimotope by one of theabove mentioned cleavage sites which enables the peptide mimotope to bemade free of the maltose binding protein.

[0054] It is particularly desirable that the peptide mimotope be a partof another peptide or polypeptide, particularly an enzyme which is usedas a reporter in immunological assays. Such reporter enzymes include,but are not limited to, alkaline phosphatase or horseradish peroxidase.As shown herein by DONPEP-AP, the peptide mimotope was fused withalkaline phosphatase, a reporter enzyme commonly used in immunologicalassays. As shown in FIG. 5, the DONPEP-AP was useful as a DON competitorin immunological assays for detecting DON in wheat extracts. DONPEP-APcan be used in other immunological assays for determining whether acorn, grain, or mixed feed sample is contaminated with DON. The peptidemimotope can also be fused to other heterologous polypeptides whichfacilitate isolation or handling of the peptide mimotope inimmunological assays. An example of such a heterologous polypeptideincludes, but is not limited to, the minor coat protein g3p offilamentous phage M13. Thus, the heterologous polypeptides that can beused to make fusion polypeptides include, but is not limited to, theminor coat protein g3p of filamentous phage M13, which was used to makephage clones DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4 and DONPEP.12containing the mimotope peptide sequence therein, and alkalinephosphatase, which was used to make DONPEP-AP. Preferably, the fusionpolypeptide is produced in a recombinant bacterium or eukaryoteexpression vector as disclosed supra. for producing the peptidemimotope.

[0055] Further, the mimotope peptide, either by itself or as part of afusion polypeptide, can be chemically conjugated to a carrier protein.Carrier proteins include, but are not limited to, bovine serum albumen(BSA), and reporter enzymes which include, but are not limited to,horseradish peroxidase or alkaline phosphatase. Further, the peptidemimotope or fusion peptide comprising the peptide mimotope can bechemically conjugated to fluorescence reporter molecules which include,but are not limited to, fluorescein or R-phycoerythrin. Methods forconjugating carrier proteins, enzymes, and fluorescence reportermolecules to peptides and fusion polypeptides are well known in the art.

[0056] The peptide mimotopes, either alone, conjugated to a carrierprotein or fluorescent reporter molecule, or as a component of a fusionpolypeptide are useful as standard and conjugates in immunoassays suchas ELISAs and RIAS, which are used to determine whether a food sample iscontaminated with DON. Currently, in such immunoassays, DON, which istoxic to the user, is used as a control or as a competitor. Theimmunoassays rely upon detection techniques which require DON to beconjugated to a carrier protein or reporter enzyme. Conjugating DON to acarrier protein or reporter enzyme has been difficult and theconjugation methods which are used require chemicals that are toxic tothe user. Therefore, because the peptide mimotopes are non-toxic, thepeptide mimotopes provide a significant advantage over DON. Inparticular, they are non-toxic to the user when used as a control orcompetitor in immunoassays, they are easier to conjugate to reporterenzymes than DON and conjugation does not require toxic chemicals, andunlike DON, they can be genetically fused to various reporter enzymesand produced by fermentation or other methods in large quantitiesthereby significantly reducing the costs associated with providingimmunoassays for detecting DON. Thus, the immunoassays of the presentinvention use either the peptide mimotopes alone or the peptidemimotopes conjugated to a carrier protein or enzyme, or geneticallyfused to a reporter enzyme such as alkaline phosphatase or horseradishperoxidase, or conjugated to a reporter fluorescence molecule.

[0057] In general, the immunoassays are performed using an enzyme-linkedimmunosorbent assay (ELISA) embodiment and can be either a competitivedirect ELISA (CD-ELISA) or competitive indirect ELISA (CI-ELISA). Toperform a CD-ELISA, a microtiter plate is provided containing aplurality of wells wherein a first well or series of wells contains amonoclonal antibody against DON immobilized to the surface therein,preferably the monoclonal antibody is mAB 6F5. To prevent non-specificbinding in the subsequent steps, it is preferable that the wells betreated with a blocking agent such as a 10% solution of non-fat milk.Next, a limiting dilution series of an aliquot of the sample are mixedwith an equal volume of an appropriate dilution of the peptide mimotopeand the mixture added to the wells containing the bound monoclonalantibody. Preferably, the mimotope peptide is conjugated to a carrierprotein or is part of a fusion polypeptide. The DON in the sample andthe peptide mimotope compete for binding to the monoclonal antibody. TheELISA is incubated for a time sufficient for monoclonal antibody-DONcomplexes to form. In general, an incubation time of about an hour at atemperature between about room temperature and 37° C. Afterwards, thewells are washed to remove any unbound material. The wells are thenincubated with a labeled antibody or labeled monoclonal antibody thatbinds to the carrier protein or fusion polypeptide to form a complexwhich can be detected when the labeled monoclonal or polyclonal antibodyis conjugated to a reporter ligand such as horseradish-peroxidase oralkaline phosphatase. Preferably, the incubation is for about an hour ata temperature between about room temperature and 37° C. A detectablesignal from the reporter indicates the sample does not contain DONwhereas an absence of a signal indicates that the sample contains DONwhich had bound all of the monoclonal antibody, thereby preventing thepeptide mimotope from binding the monoclonal antibody immobilized in thewells. Alternatively, the second monoclonal or polyclonal antibody canbe conjugated to reporter ligands such as a fluorescing ligand, biotin,colored latex, colloidal gold magnetic beads, radioisotopes or the like.When the fusion polypeptide comprises a reporter enzyme such as alkalinephosphatase, the antibody-mimotope peptide complex can be detecteddirectly without the need for a labeled antibody. In either case,detection is by methods well known in the art for detecting theparticular reporter ligand.

[0058] To perform a CI-ELISA, a microtiter plate is provided containinga plurality of wells wherein a first well or series of wells containsthe peptide mimotope, the peptide mimotope conjugated to a carrierprotein, or fusion polypeptide comprising the peptide mimotope isimmobilized to the surface therein. To prevent non-specific binding inthe subsequent steps, it is preferable that the wells be treated with ablocking agent such as a 10% solution of non-fat milk. Next, a limitingdilution series of an aliquot of the sample are added to the wellscontaining the bound peptide mimotopes along with a constant amount of amonoclonal antibody, preferably the mAB 6F5 monoclonal antibody. The DONin the sample and the peptide mimotope bound to the well surfacescompete for binding to the monoclonal antibody. The ELISA is incubatedfor a time sufficient for antibody-DON complexes to form. In general, anincubation time of about an hour at a temperature between about roomtemperature and 37° C. Afterwards, the wells are washed to remove anyunbound material. The amount of monoclonal antibody that is bound to theimmobilized mimotope peptides in the well is determined by incubatingthe wells with a labeled antibody or labeled monoclonal antibody thatbinds to the monoclonal antibody to form a complex that can be detectedwhen the labeled monoclonal or polyclonal antibody is conjugated to areporter ligand such as horseradish-peroxidase or alkaline phosphatase.Preferably for about an hour at a temperature between about roomtemperature and 37° C. A detectable signal from the reporter indicatesthe sample does not contain DON whereas an absence of a signal indicatesthat the sample contains DON which had bound all of the monoclonalantibody, thereby preventing the monoclonal antibody from binding thepeptide mimotope immobilized in the wells. The intensity of the signalprovides an estimate of the relative concentration of DON in the sample.Alternatively, the second monoclonal or polyclonal antibody can beconjugated to reporter ligands such as a fluorescing ligand, biotin,colored latex, colloidal gold magnetic beads, radioisotopes or the like.In an alternative further, the monoclonal antibody can be labeled with areporter in which case the bound monoclonal antibody can be detecteddirectly without the need for a labeled antibody. In either case,detection is by methods well known in the art for detecting theparticular reporter ligand.

[0059] Instead of an ELISA, the peptide mimotopes can be used in a radioimmunoassay (RIA) for detecting DON in a sample. The RIA procedureinvolves incubation of a monoclonal antibody against the DON, preferablymAB 6F5, simultaneously with a solution of unknown sample or knownstandard, and a constant amount of radioactively labeled peptidemimotope or fusion polypeptide. After separation of the free peptidemimotope or fusion polypeptide from bound peptide mimotope or fusionpolypeptide, the radioactivity in the respective fractions isdetermined. The concentration of DON in the unknown sample is determinedby comparing results to a standard curve. Several known methods,including the ammonium sulfate precipitation method, double antibodytechnique, solid phase RIA method in which immunoglobulin G (IgG) isconjugated to CNBr-activated SEPHAROSE gel (Pharmacia Biotech,Piscataway, N.J.), a dextran-coated charcoal Column and albumen-coatedcharcoal, is used for the separation of free from bound peptide mimotopeor fusion polypeptide in the RIA. Radioactivity is determined in aliquid scintillation counter in 5 ml of AQUASOL (a product of NewEngland Nuclear Corp., Boston, Mass.) for aqueous solutions.

[0060] In a further embodiment of the present invention, the peptidemimotope or fusion polypeptide is coupled to an energy donor fluorophoreand the monoclonal antibody is coupled to an energy acceptor or quencherfluorophore. The quencher fluorophore is attached to a monoclonalantibody against DON, preferably mAB 6F5, such that when the peptidemimotope or fusion polypeptide binds the antibody, the quencher andreporter dye are in close proximity and the reporter dye is preventedfrom fluorescing. Therefore, when a sample does not contain DON, all ofthe peptide mimotope or fusion polypeptide is bound by the monoclonalantibody. Since the quencher and reporter dyes are in close proximity,the quencher prevents the reporter dye from fluorescing. However, when asample contains DON, the DON competes with the peptide mimotope offusion polypeptide for the antibody, which results in some peptidemimotope or fusion polypeptide molecules remaining unbound. Becausethese unbound peptide mimotope or fusion polypeptide molecules are nolonger in close proximity to the quencher on the antibody, the reporterdye on these molecules will fluoresce. The intensity of fluorescence isdirectly proportional to the amount of DON in the sample. Fluorescencecan be detected with an ABA Prism Sequence Detector or TAQMAN LS-50B PCRDetection System (both available from Perkin-Elmer Applied Biosystems),or other detector that is used to detect fluorescence. Alternatively,the peptide mimotope or fusion polypeptide is coupled to an energyacceptor fluorophore and the monoclonal antibody is coupled to theenergy transfer fluorophore. The result would be the same. Preferably,the fluorophore is selected from the group consisting of fluorescein,5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine(R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxyl-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo) benzoic acid(DABCYL), tetrachloro-6-carboxyl-fluorescein (TET), VIC, and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Thisembodiment can be performed in a small reaction volume, does not need torely on microtiter plates, and enables the assay results to be knowninstantaneously.

[0061] Since the ability to test samples in the field for DONcontamination is very important, the method of the present inventionfurther includes rapid immunodiffusion based methods and apparatuses fordetecting DON in a sample. For example, a device containing the peptidemimotope, either as the peptide or as a fusion polypeptide isirreversibly fixed to a solid support. A solution containing the testsample is admixed with a solution containing a monoclonal antibodyagainst DON, preferably mAB 6F5, conjugated to a reporter and a solutioncontaining the solid support containing the mimotope. The admixture isthen applied to a porous sheet material incorporating a chromogen and asubstrate for the reporter, while keeping the support containing themimotope out of contact with the porous sheet material. If the samplecontains DON, the DON complexes with the antibody conjugate, whichcauses a color reaction on the porous sheet material. If the sample doesnot contain DON, the antibody is completely bound by the peptidemimotope or fusion polypeptide on the solid support. Since there is noantibody in solution, there is no color reaction. This method isdisclosed in U.S. Pat. No. 5,846,745 to Christensen et al. The presentinvention can be used solid phase immunodiffusion assays such as thosedisclosed in U.S. Pat. No. 5,169,789 to Berstein. The above methods areprovided as examples thus, other rapid immunoassays which are well knownin the art are also within the scope of the present invention.

[0062] Thus, the present invention can be provided as a kit thatcomprises any one of the methods described above or in U.S. Pat. No.5,620,845 to Gould et al., U.S. Pat. No. 5,559,041 to Kang et al., U.S.Pat. No. 5,656,448 to Kang et al., U.S. Pat. No. 5,728,587 to Kang etal., U.S. Pat. No. 5,695,928 to Stewart et al., U.S. Pat. No. 5,169,789to Bernstein et al. U.S. Pat. No. 4,486,530 to David et al., and U.S.Pat. No. 4,786,589 to Rounds et al. While the aforementioned discloseparticular rapid immunodiffusion methods, the present invention is notto be construed to be limited to the aforementioned. It is within thescope of the present invention to embrace derivations and modificationsof the aforementioned.

[0063] When the peptide mimotopes are conjugated to an appropriatecompound or chemical that facilitates entry of the peptide mimotopesinto the cell of the host, the peptide mimotopes can be used as atreatment for plants, animals or people exposed to DON. Alternatively,the peptide mimotope can be a part of a peptide or polypeptide, i.e.,fusion polypeptide or polypeptide, that facilitates entry of the peptidemimotope into the cell. The peptide mimotope in any of theaforementioned forms can be administered either topically, orally, or byinjection.

[0064] The present invention further includes transgenic plants thatexpress the peptide mimotope, either as an isolated peptide or as a partof a fusion peptide or polypeptide which renders the plant resistant tothe effects of DON. For example, the Fusarium fungi produce a broadrange of plant diseases such as seedling and head blight on small grainssuch as wheat and rye, ear and stalk blight on corn (maize), stem rot ofcarnation, and seedling blight and root rot of a number of other plantspecies, including, beans, clover, peanuts, and tomato. Therefore, thepresent invention provides a method for making transgenic plantsresistant to DON produced by Fusarium fungi. Since the peptide mimotopecompetes with DON for binding to the 60S ribosome but does not haveDON's inhibitory effect, transgenic plants expressing the presentinvention are resistant to the effects of DON. Transgenic plants thatexpress the peptide mimotope, either as an isolated peptide or as partof a fusion peptide or polypeptide, include, but are not limited to,wheat, rye, corn, carnation, beans, clover, and tomato.

[0065] Therefore, the transgenic plant of the present inventioncomprises a nucleic acid that encodes a peptide mimotope which has theamino acid sequence SWGPX₁PX₂ (SEQ ID NO: 6) wherein ₁X and ₂X is eachany amino acid or analog thereof, preferably wherein X₁ is an amino acidor analog thereof which has a side chain that is a hydrogen or alkyl,most preferably, wherein X₁ is L, F, or analog thereof. In particular,the amino acid sequence which is set forth in SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO: 5. In a preferred embodiment, the peptide mimotope isencoded by a nucleic acid comprising the nucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3. As disclosedsupra., the nucleic sequence encoding the peptide mimotope can becovalently linked in frame with a sequence encoding a heterologouspeptide or polypeptide.

[0066] Expression of the peptide mimotope, or peptide or polypeptidecomprising the peptide mimotope, in the plant cell requires the nucleicacid encoding the mimotope be operably linked to a transcriptionpromoter that is functional in plant cells. Examples of promoters whichare useful are viral promoters such as the cauliflower mosaic virus 35Spromoter, heat shock protein promoters such as the HSP70 promoter, lightinduced promoters such as the ST-Ls1 or the rubisco small subunitpromoter, stress response promoters such as the PR promoter, theAgrobacterium tumefaciens nos promoter, and various organ, root, tuber,and leaf specific promoters. The DRE promoter element that is inducibleunder stress is an example of a plant promoter that responds toenvironmental conditions (Yamaguchi-Shinozaki et al., Plant Cell 6:251-264 (1994)). The nucleic acid encoding the peptide mimotope ispreferably operably linked at the 3′ end to a transcription terminationsignal. An example of such a sequence is the transcription terminationsignal of the octopine synthase gene.

[0067] There are many methods known in the art for transforming a plantcell with heterologous nucleic acids. Common methods includetransformation with T-DNA containing the DNA of interest and usingAgrobacterium tumefaciens as the means for transformation or with Ti orRi plasmids using the bacterium A. rhizogenes as the means fortransformation. A suitable plasmid for transformations is the pART27/7plasmid vector isolated from Agrobacterium tumefaciens. Other methodsfor transforming a plant cell include cell fusion, electroporation,biolistic or conventional injection.

[0068] Agrobacterium related methods require special plasmid vectorssuch as intermediate or binary vectors. Intermediate vectors requireintegration into Ti or Ri plasmids by homologous recombination into theregion containing the T-DNA. The intermediate vector is transferred intothe Agrobacterium by means of conjugation in the presence of a helperplasmid. The transformed Agrobacterium is then used to transform thecell. The preferred method for transforming Agrobacterium is usingplasmids of the binary type. Binary vectors replicate both inEscherichia coli and Agrobacterium. Therefore, these vectors containingthe desired DNA can be constructed using conventional molecular biologytechniques and the recombinant plasmid directly transferred toAgrobacterium. Binary vectors usually contain a marker gene and apolylinker for inserting the desired DNA flanked by the left and rightT-DNA border regions. Both the intermediate and binary vectors containthe vir region which is necessary for transfer of the T-DNA into theplant cell.

[0069] Transformation of plant cells with transformed Agrobacterium isby co-cultivation of the cells with the transformed Agrobacterium whichresults in transfer of the T-DNA containing the desired nucleic acidinto the plant cell. Sources for plant cells are explants which caninclude but is not limited to sections of leaves, stems, roots, segmentsof petioles, flowers and flower parts, and cotyledon tissue. Wholeplants are regenerated from the infected plant material or fromprotoplasts or suspension-cultivated cells in a suitable medium whichcan contain antibiotics or biocides (e.g., kanamycin, bleomycin,hygromycin, chloramphenicol) for selection of the transformed plantcells. The ability and efficiency of regenerating a transformed ortransgenic plant using transformed isolated cells or explants isdependent on the species of plant and the type of transformed cell.Transformation of plants can be achieved according to theAgrobacterium-mediated method disclosed in U.S. Pat. No. 5,684,238 toAusich et al and U.S. Pat. No. 5,618,988 to Hauptmann et al.

[0070] Non-Agrobacterium mediated transformation such aselectroporation, injection, cell fusion, or particle bombardment do notrequire special plasmids and, therefore, can use standard plasmids suchas the pUC derivatives and conventional cloning techniques. For example,to make the transgenic plants of the present invention using theBiolistic bombardment method, plant tissue is transformed using theBiolistic method described in U.S. Pat. No. 5,767,368 to Zhong et al.Further examples of the Biolistic bombardment method are disclosed U.S.Pat. No. 5,736,369 to Bowen et al.

[0071] The following examples are intended to promote a furtherunderstanding of the present invention.

EXAMPLE 1

[0072] Identification of peptide mimotopes which bind to mycotoxinDON-specific monoclonal antibody. Monoclonal antibody 6F5 (mAB 6F5) wasused to select for peptides that mimic the mycotoxin from a library offilamentous phages that have random 7-mer peptides on their surfaces.

[0073] A phage-display heptapeptide library containing 2×10⁹ independentclones that express random peptide 7-mers fused to minor coat proteing3p of filamentous coliphage M13 was purchased from New England Biolabs,Inc. Beverly, Mass. The library had sufficient complexity to containmost if not all of the 20⁷=1.28×⁹10 possible 7-mer sequences. Afteramplification in Escherichia coli ER2537 (purchased from New EnglandBiolabs, Inc.), the phage in the library were selected by panningelution as below.

[0074] One hundred microliters of a preparation containing mAB 6F5(prepared as shown in Casale et al., Op. cit.) at 15 μg/ml in 0.01 Mphosphate-buffered saline (PBS) (pH 7.4) was dispensed into each well ofdisposable Immuno-4 microtiter strips (Dynatech Laboratories, Inc.,Chantilly, Va.). The antibody was dried overnight onto the wells in aforced-air oven at 40° C. Blank wells were coated with an equalconcentration of mouse IgG. The wells were washed 4 times by fillingeach well with 300 μl PBS and aspirating the contents. Nonspecificbinding was blocked by incubating 320 μl of 10% non-fat dry milkdissolved in PBS (10% milk-PBS) in each well for 1 hour at 37° C. andthen washing the wells four times with PBS. For panning-elutionselection, the recombinant phage display peptide library, which had beendiluted with 10% milk-PBS to about 10¹⁰ PFU/ml, was added to the wells(100 μl/well) and the wells incubated at 37° C. for 1 hour. Afterwards,the wells were washed 20 times by filling each well with about 300 μl ofPBS containing 0.1% Tween 20 (PBS-T), followed by incubation with 300 μlof PBS per well at 37° C. with shaking at 150 rpm for 1 hour. The wellswere then washed 10 times with about 300 μl PBS-T per wash. To elute thebound phage, 100 μl of DON (100 μg/ml in PBS containing 1% methanol) orPBS containing 1% methanol was added to each well, and incubated at 37°C. with shaking at 150 rpm for 1 hour. The eluted phage were collectedfrom the microtiter wells and used to infect E. coli ER2537 for phagetiter and amplification experiments. The amplified phage, whichcontained about 8 ηg/ml of DON, was used for a subsequent round ofpanning-elution selection. After four rounds of panning-elutionselection, individual plaques were picked from Luria-Bertani plates andused to infect E. coli ER2537 cells in phage production experiments.

[0075] Binding to mAB 6F5 was determined for each individual phage cloneby ELISA. The amounts of bound phage in mAB 6F5-coated microtiter wellswere determined by incubation with 100 μl of sheep anti-M13 horseradishperoxidase (HRP) conjugate, which had been diluted 1:5,000 in 10%milk-PBS, per well at 37° C. per hour, followed by incubation with 100μl of 3,3′,5,5′-tetramethylbenzidine substrate per well at 37° C. for 15minutes. The absorbance at 450 ηm was determined after the reaction wasstopped by adding 100 μl of 10% sulfuric acid per well.

[0076] After specific binding to mAB 6F5 was confirmed by ELISA, 10 mlof recombinant phage particles (10¹¹ PFU/ml in LB medium) from eachpositive phage clone was used for single-stranded DNA isolationperformed with a QIAprep Spin M13 kit (Qiagen, Inc., Chatsworth,Calif.). The single-stranded DNA was sequenced with −28 gIII and −96gIII sequencing primers (New England Biolabs, Inc.) by using Taq cyclesequencing and dye terminator chemistry at the Michigan State UniversityDNA Sequencing Facility.

[0077] Five phage isolates were identified to bind to mAB 6F5 by ELISA.These isolates were designated DONPEP.1, DONPEP.2, DONPEP.3, DONPEP.4,and DONPEP.12. Four of the five isolates had the same nucleotidesequence which was 5-AGTTGGGGTCCTTTTCCGTTT-3 (SEQ ID NO: 1), whichencodes the peptide with the amino acid sequence SWGPFPF (SEQ ID NO: 2)while isolate DONPEP.12 had the nucleotide sequence5′-TCTTGGGGTCCGCTTCCTTTT-3′ (SEQ ID NO: 3), which encodes the peptidewith the amino acid sequence SWGPLPF (SEQ ID NO: 4).

EXAMPLE 2

[0078] This example demonstrates that DON mimotope peptides actuallymimicked the epitope recognized by mAB 6F5 and was not nonspecificallybound to the surface of the antibody molecule outside the antigenbinding site. Competitive ELISAs were used to show that both phageisolates encoding either peptide competed with DON for the antigenbinding site.

[0079] To perform a competitive direct ELISA (CD-ELISA) with thephage-displayed peptide, Immuno-4 microtiter wells were coated with mAB6F5 and blocked as described in Example 1. Various concentrations of DON(0 to 10,000 ηg/ml in 1% methanol-PBS) were mixed with equal volumes ofphage-displayed peptide (diluted 1:10 in 10% milk-PBS). The mixtureswere added to mAB 6F5-coated microtiter wells (100 μl/well), and thepreparations were incubated at 37° C. for 1 hour. Afterwards, the wellswere washed 6 times with PBS-T (about 300 μl/well) and then amounts ofbound recombinant phage were determined by incubating the preparationswith 100 μl of sheep anti-M13 HRP conjugate (diluted 1:5,000 in 10%milk-PBS) per well at 37° C. for 1 hour. The amounts of bound enzymewere determined as described in Example 1. For comparison, 50 μl ofDON-HRP per well was also mixed with 50 μl of DON at variousconcentration (0 to 10,000 ηg/ml in 1% methanol-PBS) per well, and thepreparations were incubated in mAB 6F5-coated microtiter wells at 37° C.for 1 hour. The amounts of bound enzyme were determined as above.

[0080] To perform a competitive indirect ELISA (CI-ELISA) withphage-displayed peptide, 100 μl of phage-displayed peptide was dispensedinto each well of disposable Immuno-4 microtiter strips, and the peptidewas dried onto the wells in a forced air oven at 40° C. overnight. Thestrips were washed and blocked as described for the CD-ELISA. Variousconcentrations of DON (0 to 10,000 ηg/ml in 1% methanol-PBS), 50μl/well, was added to the wells, and then 50 μl of anti-DON mAB 6F5 (10μg/ml in 10% milk-PBS) was added to each well. The wells were incubatedat 37° C. for 1 hour. Then the wells were washed six times with PBS-T,and the amounts of bound anti-DON mAB 6F5 were determined by incubationwith goat anti-mouse IgG-HRP conjugate (diluted 1:2,000 with 10%milk-PBS) at 37° C. for 1 hour. The amounts of bound enzyme weredetermined as described above.

[0081] The results are shown in FIG. 2. The binding of phage clonesDONPEP.2 and DONPEP.12 to immobilized mAB 6F5 was competitivelyinhibited by free DON. This strongly suggested that these two phageclones bind to the antigen binding site of the mAB, mimicking, in part,the structural epitope of DON.

EXAMPLE 3

[0082] This example demonstrates that a synthetic peptide, C430, whichcomprises the amino acid sequence SWGPFP (SEQ ID NO: 2), was alone wassufficient for binding to the mAB, independent of the phage structuralcontext.

[0083] C430, a DON peptide mimotope with a structurally flexible linkerand a cysteine residue, which has the sequence NH₂-SWGPFPFGGGSC-COOH(SEQ ID NO: 5) was synthesized via N-(9-fluorenylmethoxycarbonyl) (Fmoc)chemistry at Bio-Synthesis, Inc. of Lewisville, Tex. C430 was used atvarious concentrations to compete with DON-HRP for binding to mAB 6F5 ina CD-ELISA. C430 was also conjugated to HRP with a sulfo-MBScross-linker. The procedure used for the CD-ELISA performed with theC430-HRP conjugate was the same as the procedure used for the ELISAperformed with DON-HRP described above, except that C430-HRP (diluted1:5,000 in blocking buffer) replaced the DON-HRP.

[0084] The results shown in FIG. 3A show that binding of DON-HRP toimmobilized mAB 6F5 was inhibited by free C430, and the results shown inFIG. 3B show that binding of C430-HRP conjugate was inhibited by freeDON. This indicates that the peptide alone was sufficient for binding tothe mAB, independent of the phage structural context. When C430 was usedto compete with DON-HRP or C430-HRP conjugate for binding to mAB 6F5,the 50% inhibitory concentrations were 0.64 to 0.8 μM, whereas 3.4 μMfree DON was required to obtain 50% inhibition of DON-HRP or C430-HRPconjugate binding to the same mAB. This indicates that mAB 6F5 has ahigher affinity for C430 than for DON. In a similar CD-ELISA, none ofthe individual amino acids in C430 (at concentrations up to 34 μM)significantly inhibited binding to DON-HRP. This suggests that thesequence in C430 was important for specific binding to mAB 6F5, sinceindividual amino acids did not bind mAB 6F5 specifically (data notshown).

EXAMPLE 4

[0085] This example demonstrates that the DON mimotope peptide isstructurally stable in a protein context different from the phageprotein context or as a free peptide.

[0086] A 179-bp DNA fragment encoding the g3p signal peptide, DONPEP.2,and the first 17 amino acids of M13 phage g3p protein from the DONPEP.2phage isolate was amplified by polymerase chain reaction (PCR) performedwith Pfu DNA polymerase, a sense primer,5-GCCAAGCTTAGATCTTGGAGCCTTTTTTTTGGAG-3′ (SEQ ID NO: 7), and an antisenseprimer, 5′-CCGGTCGACCTGTATGGGATTTTGCTAAACAACT-3′ (SEQ ID NO: 8). Aftergel purification, the amplified DNA was digested with BglII and SalI andwas cloned into BamHI-SalI-digested pLIPS (Carrier et al., J. Immunol.Methods 181: 177-186 (1995)), which generated plasmid pQY7 containingthe nucleic acid sequence encoding DONPEP.2 covalently linked to the 5′end of the nucleic acid sequence encoding alkaline phosphatase. Theligated product was used to transform E. coli DH11S competent cells(GIBCO BRL, Gaithersburg, Md.), which generated DH11S/pQY7.DONPEP-alkaline phosphatase (AP) fusion protein was produced fromDH11S/pQY7 grown in SB medium (35 g bactotrypase, 20 g bacto yeastextract, 5 g NaCl per liter) containing 100 μg/ml ampicillin and inducedby 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The periplasmicDONPEP-AP fusion protein was extracted by suspending the bacterial cells(1:5, vol:vol) in lysis buffer (50 mM Tris-HCl [pH 8.0], 20% sucrose, 10mM EDTA, 0.1 mg/ml lysozyme, 0.5 mM phenylmethylsulfonyl fluoride).After 1 hour on ice with agitation, the preparation was centrifuged at7,000× g for 30 minutes. The supernatant fraction was filtered through a0.4 μm-pore-size porous filter. The ability of the DONPEP-AP fusionpeptide to compete with DON for binding to mAB 6F5 was determined byCD-ELISA performed as previously described. Briefly, Immuno-4 wells werecoated with mAB 6F5 and washed as described above. Serial dilutions ofDON (0 to 10,000 ηg/ml in 0.05 M Tris-buffered saline pH 7.4 (TBS) wereeach mixed with equal volumes of DONPEP-AP fusion protein (periplasmicextract diluted 1:7 with 2% nonfat dry milk-TBS). The mixtures (100μl/well) were added to the mAB 6F5 coated microtiter wells and incubatedat 37° C. for 1 hour. After the wells were washed six times with 300 μlof TBS containing 0.1% Tween 20, the amounts of bound DONPEP-AP for eachmixture were determined by incubation with a p-nitrophenyl phosphatesubstrate solution at 37° C. for 45 minutes. The absorbance at 405 nmwas determined.

[0087] The results, which are shown in FIG. 4, show that the DONPEP-APfusion protein had alkaline phosphatase activity, but more importantly,the results show that its specific binding to mAB 6F5 was similar tothat of DON-HRP. This indicates that the DON peptide mimotope sequenceis structurally stable in a different protein structural context.

EXAMPLE 5

[0088] This example demonstrates the feasibility of using DON mimotopepeptide sequence as immunochemical reagents for DON immunoassays in foodand feed.

[0089] A CD-ELISA was performed as above using wheat extracts spikedwith DON. As shown in FIG. 5, both the C430-HRP conjugate and DONPEP-APfusion protein exhibited binding to immobilized mAB 6F5 in the wheatextract which was similar to the binding of DON-HRP. All three HRPconjugates produced similar linear inhibition curves at DONconcentrations ranging from 0.1 to 10 μg/ml in wheat extract. However, aslightly lower level of absorbance was observed in the CD-ELISAperformed with C430-HRP and DONPEP-AP in wheat extract than in PBSbuffer, indicating that the wheat extract interfered with binding of thepeptide mimotope to anti-DON mAB 6F5 to some extent.

EXAMPLE 6

[0090] This example was performed to determine whether the DON peptidemimotope sequence can elicit an immune response in animals similar tothat of DON and, therefore, be useful as an alternative antigen for DON.

[0091] Synthetic peptide C430 was conjugated to BSA with am-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS)cross-linker. The C430-BSA conjugate, at a molar ratio of peptide C430to the carrier protein BSA of about 29:1, was used to inject 7-week-oldBALB/c female mice intraperitoneally or 6-month-old New Zealand whiterabbits subcutaneously. The initial injections used for the micecontained 100 to 200 μg of C430-BSA conjugate in 200 μl ofsaline-Freund's complete adjuvant (1:1). These injections were followedat 3-week intervals by booster injections consisting of 100 to 200 μg ofconjugate in 200 μl of saline-Freund's incomplete adjuvant (1:1). Theinitial inoculum used for rabbits consisted of 1 mg of the C4430-BSAconjugate in 1 ml of saline Freund's complete adjuvant (1:1) followed bybooster injections at 4-week intervals consisting of 250 μg of conjugatein 1 ml of saline-Freund's incomplete adjuvant. The animals were bled 1week after each booster injection. The antisera were screened forspecific binding in phage-displayed DONPEP.2 coated wells by CI-ELISA asdescribed above in which C430 or DON was used as the complete adjuvant.

[0092] Both the mice and rabbits produced antibody specific to the DONpeptide mimotope sequence after the second injection of C430-BSAconjugate. FIG. 6 shows that after the fourth injection, the anti-serumexhibited a strong antibody response against the DON peptide mimotopesequence. A synthetic peptide C430 concentration of 0.39 μM resulted in50% inhibition of antiserum binding to the immobilized phage-displayedpeptide. However, binding of antisera to immobilized phage-displayedpeptide was not inhibited by free DON in solution, indicating that theantibodies in the immunized animals were not specific for DON. Thus, thepeptide mimotope sequences do not represent a true image of the DONsurface structure.

EXAMPLE 7

[0093] This example was to determine whether the DON peptide mimotopesequence has any cytotoxic effect. It has been known that one of theeffects of DON is to cause cytotoxicity through cell apoptosis andwhether the peptide mimotope sequences were like DON and had an effecton new protein synthesis.

[0094] Comparisons of the cytotoxic effect by the peptide mimotopesequences to the effects by DON were performed on mouse bone marrowcells. Ten-week-old B6C3F1 mice were euthanized by cervical dislocation,and the femurs were removed. The bone marrow cells were flushed from thefemurs by using a 1-ml syringe and a 25-gauge needle. Erythrocytes werelysed with 0.83% ammonium chloride. Cell number and viability weredetermined by trypan blue dye exclusion by using a hemacytometer. Thecells were cultured at a concentration of 10⁶ cells/ml in 96-wellflat-bottom plates in RPMI-1640 in a humidified incubator containing 5%CO₂. RPMI-1640 was supplemented with 100 units penicillin per ml, 100 μgof streptomycin per ml, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1mM nonessential amino acids, 2 mM glutamine, and 10% fetal bovine serum.DON and synthetic peptide C430 were separately diluted in RPMI-1640. Thefinal concentrations of C430 were 0.34, 3.4. and 34 μM and the finalconcentration of DON was 3.4 μM. Controls consisted on RPMI-1640.Duplicate cultures were treated. After 18 hours of exposure to DON orC430, cell viability was determined by using an MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]conversion assay as described by Marin et al. in Toxicology 114: 67-79(1996).

[0095] As expected, DON at a concentration of 3.4 μM caused 40 to 60%cell death after 18 hours of incubation. In contrast, synthetic peptideC430 did not have any adverse effect on the viability of the bone marrowcells at any of the concentrations tested after 18 hours incubation.When combined with DON, C430 at any of the above test concentrations didnot significantly increase or decrease the cell viability caused by DON.This indicates that there was no significant synergism or antagonismbetween the DON peptide mimotope and DON with respect to bone marrowcell death.

EXAMPLE 8

[0096] This example was to determine whether the DON peptide mimotopesequence were like DON and had an effect on new protein synthesis.

[0097] To determine the effects on protein synthesis, in vitrotranslation assays were performed using γ-globulin mRNA template. DON orsynthetic peptide C430 (0 or 3.4 μM in a 50-μl (final volume) reactionmixture) was added to 30 μl of a biotin translation mixture (BoehringerMannheim Corp., Indianapolis, Ind.) containing reticulocyte lysate, 10pmol of biotin-lysine-tRNA^(lys), 42 μM amino acids (without lysine),spermidine, energy mixture, dithiothreitol, 83 mM potassium acetate, and83 mM magnesium acetate. The mixtures (46 μl ) were incubated on ice for10 minutes, and then 4 μl of γ-globulin mRNA (0.5 μg/ml) was added. Thefinal reaction mixture (50 μl) was incubated at 30° C. for 1 hour. Thetranslated protein samples were stored at −80° C. before analysis byWestern blot.

[0098] For Western blot analysis, 3 μl aliquots of protein from the invitro translation assays were boiled for 10 minutes after mixing withsodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis loadingbuffer. The samples were then loaded into the wells of a mini SDS-10%polyacrylamide gel and electrophoresed at 80 volts for 2 hours. The invitro synthesized proteins were detected by electrotransfer to apolyvinylidene difluoride transfer membrane (DuPont NEN ResearchProducts, Boston, Mass.) followed by incubation withstrepavidin-peroxidase conjugate (Boehringer Mannheim Corp.). Boundenzyme was visualized by incubation with SUPERSIGNAL ULTRAchemiluminescent substrate (Pierce Chemical Co., Rockford, Ill.) and byexposing to Kodak XAR5 autoradiography film (Kodak Corp., Rochester,N.Y.).

[0099] As shown in FIG. 7, DON at a concentration of 3.4 μMsignificantly inhibited new protein synthesis whereas 3.4 μM of C430 hadno such inhibitory effect. Unexpectedly, when 3.4 μM DON was mixed withan equimolar amount of C430, new protein synthesis was not inhibited.This indicates that at least in vitro, C430 has an antagonistic to theinhibitory effect of DON on protein synthesis.

EXAMPLE 9

[0100] This example provides a structural model of the DON peptidemimotope sequence and compares the structural model with the structureof DON.

[0101] To do homologous sequence searching and peptide modeling, thecomputer program Sequery (Collawn et al., EMBO J. 10: 3247-3252 (1991);Craig et al., J. Molec. Biol. 281: 183-201 (1998)) was used to searchfor amino acid sequences similar to the DONPEP.2 sequence (SWGPFPF) in adatabase of nonhomologous protein structures derived from the ≦25%identity set of the Protein Data Bank (PDB) Select list. Sequeryidentified all occurrences of protein sequences in the PDB that matchedthe tetrapeptidyl fragments of DONPEP.2. The following conserved aminoacid residue substitutions were allowed during the sequence search: Sfor T, W for F, P for ST, and F for ST. The resulting sequence analogsconsisted of tetrapeptides that matched a portion of DONPEP.2 and hadknown three-dimensional structures which were available in theBrookhaven PDB. The secondary structures of these sequence analogs weresubsequently analyzed using the Superpositional Structural Assignmentcomputer program (Craig et al., J. Molec. Biol. 281: 183-201 (1998)).This program superimposed the sequence analogs onto a set of regularsecondary structure templates and assigned each sequence analog to thestructural category, e.g., α-helix, β-strand, reverse turn, whichmatched most closely within a 1.0-Å main-chain root-mean-squarepositional deviation (RSMD); if no template matched a 1.0-Å RSMD, thestructure of the sequence analog was considered irregular. The sequenceanalogs that were found to be irregular were analyzed visually by usingmolecular graphics, so that slightly irregular structures, e.g., a bentα-helical turn, could be assigned to the most appropriate structuralcategory.

[0102] A three-dimensional structural model of DONPEP.2 was createdbased on the structural sequence analogs by using the molecular modelingsoftware Insight II (available from Molecular Simulations Inc., SanDiego, Calif.) and a Silicon Graphics Indigo² Extreme computer (MontainView, Calif.). The overlapping amino acid residues of the sequenceanalogs were superimposed, and a consensus structure was created, whichconsisted of the peptide main-chain atoms and proline side chains. Sidechains for the nonproline residues were added to the model by using therotamer library of Insight II. Final side chain positions were selectedbased on consensus between the sequence analogs and the absence ofsteric overlap. Interactions within the model were then optimized byusing 100 steps of backbone-restrained steepest descent energyminimization with the cff91 force filed of the Discover 3 module ofInsight II. The stereochemical quality of the model was validated withProcheck (Laskowski et al., J. Appl. Cryst. 26: 283-291 (1993)).

[0103] The structural and chemical similarities between DON and DONPEP.2peptide model were analyzed using PowerFit (Microsimulations Inc.,Mahwah, N.J.), which tests a large number of random three-dimensionalalignments of two structures by using a Monte Carlo search algorithm.The quality of the superpositions was scored by using a complementaryfunction measuring van der Waals and electrostatic overlap between thetwo structures. The three-dimensional structure of nivalenol (CambridgeStructural Database (CSD) code: DUTJOR10), which was obtained from theCSD (http://www.ccdc.cam.ac.uk), was used in place of DON for thesuperposition because a three-dimensional structure for DON is notavailable. Nivalenol binds to mAB 6F5 with slightly less affinity thanDON binds to mAB 6F5. The side chain angles of the S, W, and F aminoacid residues in the peptide were allowed to rotate during PowerFitsuperposition, and only superpositions with the stereochemicallyacceptable side chain conformation were accepted. No bonds in the rigidnivalenol structure were allowed to rotate. The results of the alignmentwere analyzed visually with Insight II.

[0104] Within the set of nonhomologous protein structures, 31 analogs ofDONPEP.2 tetrapeptides were found. The sequence search was limited tofour residues because close sequence matches to more than four residuesare rarely found in the Brookhaven PDB. The secondary structures ofthese tetrapeptides, which were assigned based on close backbonesuperposition with ideal secondary structures (α-helices, β-strands,reverse turns), were predominantly in the β-strand configuration (Table1).

[0105] To construct a three-dimensional structural model of SWGPFPF fromtetramer analogs in Table 1, we began by superimposing the analogs forresidues 1 to 4 (SWGP) were superimposed, which all formed a β-strand.The superpositions for residues 2 to 5 (WGPF) and 3 to 6 (GPFP)indicated that the central region of the peptide could for either aβ-strand or a reverse turn. However, the pair of proline residues in thesequence resulted in a significant kink even in the β-strand-likeanalogs (all of which had a superpositional RMSD equal to or greaterthan 0.9 Å in the GPFP region). Similarly, in the turn-forming analogs,the two proline residues made it impossible to form an ideal reverseturn. Thus, it seems likely that the central region forms a loose,inverted U-shaped turn flanked by residues in the β-strand conformation,which is prevalent in the analogs for residues 4 to 7 (shown in Table1). TABLE 1 Secondary structure assignment of DONPEP.2 sequenceanalogs*. Confor- Backbone PDB Residue Analog mation RMSD (Å) Code ChainRange SWGP DON mimotope peptide 1-4 SWGS Strand 0.580 Penicillinamidohydrolase 1pnk B 64-67 SWGT Strand 1.264 Sulfhydryl proteinase 9pap176-179 SWGT Strand 0.946 Xylanase 1xnb 84-87 TWGP Strand 0.794Bluetongue virus coat protein 1bvp 1 118-121 SFGT Strand 0.734Creatinase 1chm A 325-328 SFGT Strand 0.453 Phosphotransferase 3pmg A377-380 WGPF DON mimotope peptide 2-5 FGSF Turn 0.897 Fe(III) superoxidedismutase 1isc A 100-103 FGSY Strand 0.465 Beta-lactamase 2blt A 322-325FGSY Strand 0.730 Flavodoxin 4fxn 85-88 FGTY Strand 0.530 Simian virus40 coat protein 1sva 1 221-224 WGTY Turn 0.412 Xylanase 1xnb 85-88 FGPWStrand 0.956 Vitelline membrane protein 1 1vmo A 126-129 WGTW Turn 0.820Glycosyl transferase 1bpl B 215-218 WGTW Strand 0.660 Vitelline membraneprotein 1 1vmo A 70-73 GPFP DON mimotope peptide 3-6 GPFP Strand 1.080Adenylosuccinate synthetase 1ade A 276-279 GPFP Turn 0.821 N-cadherin1nch A 15-18 GPFP Turn 0.518 Photosynthetic reaction center 1pcr H 54-57GPFT Turn 0.966 Aconitase 8acn 324-327 GPFT Turn 0.702 Monellin 1mol A 9-12 GPYP Strand 0.909 Dioxygenase 2pcd M 445-448 GTFP Strand 1.110Black beetle virus coat protein 2bbv C 131-134 GTFP Turn 0.732 Glycosyltransferase 1xyz A 806-809 PFPF DON mimotope peptide 4-7 PFSF Strand1.119 N-acethlneuraminate lyase 1nal 1 112-115 PFSY Turn 0.888 Acidphosphatase 1kbp A 218-221 PFTY Strand 1.127 Dialkyglycine decarboxylase2dkb 170-173 PYSY Turn 0.528 Transthyretin 1ttb A 113-116 PYTF Strand0.599 Dethiobiotin synthase 1dts 73-76 PYTF Strand 0.498 Zincendopeptidase 1iae 16-19 PYTF Strand 0.554 Acid phosphatase 1kbp A127-130 TYPY Turn 0.952 Adenylosuccinate synthetase 1ade A 234-237 TYPYStrand 1.086 Sulfhydryl proteinase 9pap 85-88

[0106] The conformation of the turn shown in FIG. 1C is based onsuperposition of the turn forming analogs for residues 2 to 5.

[0107] The similarity between DON and the SWGPFPF model was determinedby using POWERFIT to evaluate favorable three-dimensional superpositionof nivalenol (FIGS. 1B and 1D), a close analog of DON (FIG. 1A), ontothe peptide model. Ten independent superpositions, beginning with randomconformations, were performed by using POWERFIT. Three of the tenstructures with low superpositional energies showed a preference for theDON analog to align in a specific position along the peptide backbone ofthe model in the region spanning from the second-residue (tryptophan)main-chain nitrogen to the fifth-residue (phenylalanine) carbonyl carbon(FIG. 1E). The remaining superpositions showed a preference fornivalenol to align along the peptide backbone as well, although italigned with shorter sections. Also, side chain atoms of the secondresidue (tryptophan) in SWGPFPF overlapped with atoms of nivalenol inseveral of the superposition results.

EXAMPLE 10

[0108] A radio immunoassay (RIA) is performed using the C430 as follows.The RIA procedure involves incubation of monoclonal antibody mAB 6F5simultaneously with a solution of unknown sample or known standard, anda constant amount of radioactively labled C430. After separation of thefree from bound C430, the radioactivity in the respective fractions isdetermined. The concentration of DON in the unknown sample is determinedby comparing results to a standard curve. Several known methods,including the ammonium sulfate precipitation method, double antibodytechnique, solid phase RIA method in which the immunoglobulin G (IgG) isconjugated to CNBr-activated SEPHAROSE gel (Pharmacia Biotech), adextran-coated charcoal colum and albumen-coated charcoal, is used forthe separation of free and bound C430 in RIA. A preferred method is anammonium precipitation method performed according to F. Chu et al.,Appl. Environ. Microbiol. 37: 104-108 (1979). In general, 50 μl ofradioactive C430 (10,000 to 15,000 dpm) is incubated with 0.15 ml ofanti-DON mAb solution of various dilutions in phosphate buffer (0.1 M,pH 7.2) at room temperature for 30 minutes, and then at 60° C.overnight. Separation of the bound from the free C430 is achieved by anammonium sulfate precipitation method according to Chu et al. (ibid.).Radioactivity is determined in a liquid scintillation counter in 5 ml ofAQUASOL (a product of New England Nuclear Corp., Boston, Mass.) foraqueous solutions.

EXAMPLE 11

[0109] This example provides an RIA assay for DON in a wheat sampleusing radioactively labeled C430. DON is extracted from the wheat samplewith acetonitrile:water (84:16), defatted with hexane, and reacted withacetic anhydride in pyridine to form DON-triacetate. The reactionmixture is loaded onto a C-18 cartridge to remove excess reagents andimpurities. Acetylated DON is eluted from the cartridge with 50%methanol solution, and analyzed by RIA using mAB 6F5 and radioactivelylabeled C430.

[0110] While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1 8 1 21 DNA Artificial Sequence Description of Artificial SequenceSequence encoding the DONPEP.2 peptide mimotope of deoxynivalenol 1agttggggtc cttttccgtt t 21 2 7 PRT Artificial Sequence Description ofArtificial Sequence DONPEP.2 peptide mimotope of deoxynivalenol 2 SerTrp Gly Pro Phe Pro Phe 1 5 3 21 DNA Artificial Sequence Description ofArtificial Sequence Sequence encoding the DONPEP.12 peptide mimotope ofdeoxynivalenol 3 tcttggggtc cgcttccttt t 21 4 7 PRT Artificial SequenceDescription of Artificial Sequence DONPEP.12 peptide mimotope ofdeoxynivalenol 4 Ser Trp Gly Pro Leu Pro Phe 1 5 5 12 PRT ArtificialSequence Description of Artificial Sequence C430, the DONPEP.2 with astructurally flexible linker and a cysteine residue 5 Ser Trp Gly ProPhe Pro Phe Gly Gly Gly Ser Cys 1 5 10 6 7 PRT Artificial SequenceDescription of Artificial Sequence A consensus sequence for peptidemimotopes of deoxynivalenol 6 Ser Trp Gly Pro Xaa Pro Xaa 1 5 7 34 DNAArtificial Sequence Description of Artificial Sequence Sense primer 7gccaagctta gatcttggag cctttttttt ggag 34 8 34 DNA Artificial SequenceDescription of Artificial Sequence Antisense primer 8 ccggtcgacctgtatgggat tttgctaaac aact 34

We claim:
 1. A peptide mimotope of deoxynivalenol (DON) comprising aminoacid sequence SWGPX₁PX₂ which binds to a monoclonal antibody against theDON wherein X₁ is L, F, or analog thereof and X₂ is each any amino acidor analog thereof.
 2. A peptide mimotope of deoxynivalenol (DON)comprising the amino acid sequence SWGPFPF which binds to a monoclonalantibody against the DON.
 3. A peptide mimotope of deoxynivalenol (DON)comprising the amino acid sequence SWGPLPF which binds to a monoclonalantibody against the DON.
 4. A peptide mimotope of deoxynivalenol (DON)comprising the amino acid sequence SWGPFPFGGGSC which binds to amonoclonal antibody against the DON.
 5. The peptide mimotope of any oneof claims 1, 2, 3 or 4 wherein the peptide mimotope is antagonistic tothe inhibitory effects of the DON on in vitro protein synthesis.
 6. Thepeptide mimotope of any one of claims 1, 2, 3, or 4 wherein the peptidemimotope is conjugated to a reporter for an immunological assay whereinthe reporter is selected from the group consisting of alkalinephosphatase, horseradish peroxidase, or fluorescence molecule.
 7. Thepeptide mimotope of any one of claims 1, 2, 3 or 4 wherein the peptidemimotope is a part of a peptide or polypeptide.
 8. The peptide mimotopeof claim 7 wherein the polypeptide is selected from the group consistingof alkaline phosphatase and horseradish peroxidase.
 9. A nucleic acidencoding a nucleic acid that encodes a peptide mimotope of DON, whichbinds to a monoclonal antibody against DON and is antagonistic to theinhibitory effects of DON on in vitro protein synthesis, comprising anamino acid sequence selected from the group consisting of SWGPLPF,SWGPFPF, and SWGPFPFGGGSC.
 10. A clone in a microorganism expressing apeptide mimotope of deoxynivalenol (DON) comprising amino acid sequenceSWGPX₁PX₂ which binds to a monoclonal antibody against the DON whereinX₁ is L, F, or analog thereof and X₂ is each any amino acid or analogthereof.
 11. The clone in claim 10 wherein the peptide mimotopecomprises an amino acid sequence selected from the group consisting ofSWGPFPF, SWGPLPF, and SWGPFPFGGGSC.
 12. The clone of claim 10 or 11wherein the peptide mimotope is antagonistic to the inhibitory effectsof the DON on in vitro protein synthesis.
 13. The clone of claim 10 or11 wherein the peptide mimotope is encoded by a nucleic acid in aplasmid.
 14. The clone of claim 10 or 11 wherein the peptide mimotope isencoded by a nucleic acid in a recombinant virus vector.
 15. The cloneclaim 10 or 11 wherein the microorganism expressing the peptide mimotopeis selected from the group consisting of bacteria and yeast.
 16. Theclone of claim 10 or 11 wherein the peptide mimotope is a part of apeptide or polypeptide.
 17. The peptide mimotope of claim 16 wherein thepolypeptide is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.
 18. A transgenic plantcontaining a nucleic acid that encodes a peptide mimotope ofdeoxynivalenol (DON) wherein the peptide mimotope of DON binds to amonoclonal antibody against DON and is antagonistic to the inhibitoryeffects of DON on in vitro protein synthesis.
 19. The transgenic plantof claim 18 wherein the peptide mimotope comprises amino acid sequenceSWGPX₁PX₂ wherein X₁ is L, F, or analog thereof and X₂ is each any aminoacid or analog thereof.
 20. The transgenic plant of claim 19 wherein thepeptide nimotope comprises an amino acid sequence selected from thegroup consisting of SWGPFPF, SWGPLPF, and SWGPFPFGGGSC.
 21. Thetransgenic plant of claim 18 wherein the peptide mimotope isantagonistic to the inhibitory effects of the DON on in vitro proteinsynthesis.
 22. A peptide mimotope of deoxynivalenol (DON) which competeswith DON for binding to a monoclonal antibody, is antagonistic to theinhibitory effects of the DON on in vitro protein synthesis, and doesnot elicit antibodies in mice against the DON.
 23. The peptide mimotopeof claim 22 wherein the monoclonal antibody is from hybridoma cell line6F5.
 24. In a method for determining whether a sample containsdeoxynivalenol (DON) which comprises providing monoclonal antibodyagainst the DON, reacting the monoclonal antibody with the sample in areaction mixture containing a labeled DON as a competitor, anddetermining whether the sample contains the DON, the improvementcomprises providing as the competitor a peptide mimotope of the DONwhich is bound by the monoclonal antibody.
 25. The method of claim 24wherein the peptide mimotope comprises amino acid sequence SWGPX₁PX₂wherein X₁ is L, F, or analog thereof and w is each any amino acid oranalog thereof.
 26. The method of claim 24 wherein the peptide mimotopehas an amino acid sequence selected from the group consisting ofSWGPFPF, SWGPLPF, and SWGPFPFGGGSC.
 27. The method of any one of claims24, 25, or 26 wherein the peptide mimotope is conjugated to a reporterfor an immunological assay wherein the reporter is selected from thegroup consisting of alkaline phosphatase, horseradish peroxidase, orfluorescence molecule.
 28. The method of any one of claims 24, 25, or 26wherein the peptide mimotope is a part of a peptide or polypeptide. 29.The method of claim 28 wherein the polypeptide is selected from thegroup consisting of alkaline phosphatase and horseradish peroxidase. 30.The method of claim 24 wherein the monoclonal antibody is mAB 6F5.
 31. Amethod for determining whether a sample contains deoxynivalenol (DON)which comprises: (a) incubating in a reaction the sample, a monoclonalantibody against the DON, and a peptide mimotope of the DON which is acompetitor of the DON for the monoclonal antibody; (b) detecting in thereaction a complex consisting of the DON bound by the monoclonalantibody and a complex formed by the peptide mimotope and monoclonalantibody; and (c) comparing an amount of each of the complexes wherein adecrease in the amount of the complex comprising the peptide mimotopeindicates the sample contains the DON.
 32. The method of claim 31wherein the peptide mimotope comprises amino acid sequence SWGPX₁PX₂wherein X₁ is L, F, or analog thereof and X₂ is each any amino acid oranalog thereof.
 33. The method of claim 32 wherein the peptide mimotopecomprises an amino acid sequence seleced from the group consisting ofSWGPFPF, SWGPLPF, and SWGPFPFGGGSC.
 34. The method of claim 31 whereinthe monoclonal antibody is produced by hybridoma cell line 6F5.
 35. Themethod of any one of claims 31, 32, or 33 wherein the peptide mimotopeis conjugated to a reporter for an immunological assay wherein thereporter is selected from the group consisting of alkaline phosphatase,horseradish peroxidase, or fluorescence molecule.
 36. The method of anyone of claims 31, 32, or 33 wherein the peptide mimotope is a part of apeptide or polypeptide.
 37. The method of claim 36 wherein thepolypeptide is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.
 38. A kit for determiningwhether a sample contains deoxynivalenol (DON) comprising: (a) amonoclonal antibody against the DON; (b) a peptide mimotope of the DONwhich is a competitor of the DON for binding to the monoclonal antibody;and (c) instructions for using the kit.
 39. The kit of claim 38 whereinthe peptide mimotope comprises amino acid sequence SWGPX₁PX₂ wherein X₁is L, F, or analog thereof and is each any amino acid or analog thereof.40. The kit of claim 39 wherein the peptide mimotope comprises an aminoacid sequence selected from the group consisting of SWGPFPF, SWGPLPF,and SWGPFPFGGGSC.
 41. The kit of claim 38 wherein the monoclonalantibody is produced by hybridoma cell line 6F5.
 42. The kit of any oneof claims 38, 39, or 40 wherein the peptide mimotope is conjugated to areporter for an immunological assay wherein the reporter is selectedfrom the group consisting of alkaline phosphatase, horseradishperoxidase, or fluorescence molecule.
 43. The kit of any one of claims38, 39, or 40 wherein the peptide mimotope is a part of a peptide orpolypeptide.
 44. The kit of claim 43 wherein the polypeptide is selectedfrom the group consisting of alkaline phosphatase and horseradishperoxidase.
 45. A method for making a plant resistant to dexoynivalenol(DON) comprising introducing into the plant's genome a nucleic acid thatencodes a peptide mimotope of the DON which is operably linked to atranscription promoter, wherein the peptide mimotope is antagonistic tothe inhibitory effects of DON on in vitro protein synthesis.
 46. Themethod of claim 45 wherein the nucleic acid encodes the peptide mimotopewhich comprises amino acid sequence SWGPX₁PX₂ wherein X₁ is L, F, oranalog thereof and X₂ is each any amino acid or analog thereof.
 47. Themethod of claim 45 wherein the peptide mimotope amino acid sequence isselected from the group consisting of SWGPFPF, SWGPLPF, andSWGPFPFGGGSC.
 48. The method of any one of claims 45, 46, or 47 whereinthe peptide mimotope is part of a peptide or polypeptide.
 49. Atransgenic plant comprising a nucleic acid that encodes a peptidemimotope of the DON which is operably linked to a transcriptionpromoter, wherein the peptide mimotope is antagonistic to the inhibitoryeffects of DON on in vitro protein synthesis.
 50. The transgenic plantof claim 49 wherein the nucleic acid encodes the peptide mimotope whichcomprises amino acid sequence SWGPX₁PX₂ wherein X₁ is L, F, or analogthereof and X₂ is each any amino acid or analog thereof.
 51. Thetransgenic plant of claim 50 wherein the peptide mimotope amino acidsequence is selected from the group consisting of SWGPFPF, SWGPLPF, andSWGPFPFGGGSC.
 52. The transgenic plant of any one of claims 49, 50, or51 wherein the peptide mimotope is part of a peptide or polypeptide. 53.A method for treating an organism exposed to deoxynivalenol (DON)comprising treating the organism with a peptide mimotope of the DONwhich is antagonistic to the inhibitory effects of the DON on in vitroprotein synthesis.
 54. The method of claim 53 wherein the peptidemimotope comprises amino acid sequence SWGPX₁PX₂ wherein X₁ is L, F, oranalog thereof and X₂ is each any amino acid or analog thereof.
 55. Themethod of claim 54 wherein the peptide mimotope amino acid sequence isselected from the group consisting of SWGPFPF, SWGPLPF, andSWGPFPFGGGSC.
 56. The method of any one of claims 53, 54, or 55 whereinthe peptide mimotope is part of a peptide or polypeptide.